Apoptosis is programmed cell death, a naturally occurring process involved in both the development and aging of cells. It is the process whereby the body can rid itself of unwanted, old, or damaged cells. Apoptosis is the physiological counterpart of cell proliferation. It is essential for both biological processes such as normal tissue turnover, embryonic development, and maturation of the immune system, including pathological processes, such as hormone deprivation, thermal stress and metabolic stress. (Wyllie, A. H., in Bowen and Lockshin (eds.) Cell Death in Biology and Pathology (Chapman and Hall, 1981), at 9-34).
Apoptosis is characterized by a decrease in cell volume, a condensation of chromatin, cellular budding, and the fragmentation of DNA into a ladder of 180 base pair (bp) oligomers with 3'-OH free ends, a hallmark of apoptosis. Cell membranes maintain their integrity through the process, and lysosomes remain intact. There is no inflammatory response from apoptosis. Affected cells undergo phagocytosis by adjacent normal cells and by some macrophages.
The biochemical effector pathways that underlie the apoptotic mechanisms are as yet unknown. It has been suggested that the apoptotic mechanism involves one or more Ca.sup.2+ /Mg.sup.2+ -dependent endogenous endonucleases (Arends et al., (1990) Am. J. Pathol. 136:593-608); transglutaminase activity (Fesus et al., (1987) FEBS Lett. 224:104-108; Taress et al., (1992) J. Biol. Chem. Cell 75:653-660); and the generation of oxygen radicals (Hockenberry et al., (1993) Cell 75:241-251; Butke and Sandstrom (1994) Immun. Today 15:7-10). It appears that gene expression is required for apoptosis as this process can be stopped by inhibitors of RNA or protein synthesis (Martin et al., J. Cell Biol. 106:829-844 (1988)).
Although the apoptotic mechanism has not been determined, several proto-oncogenes, including c-myo, p53, and bol-2, have been implicated in its control (See Grand et al., (1995) Exp. Cell Res. 218:439-451). The delicate balance between these genes determines whether a cell will underto apoptosis or survive. None of these gene products, however, have been identified as a specific marker for apoptosis because these genes are involved in other biochemical processes.
Apoptosis can be activated by a number of intrinsic or extrinsic signals. These signals include the following: mild physical signals, such as ionization radiation, ultraviolet radiation, or hyperthermia; low to medium doses of toxic compounds, such as azides or hydrogen peroxides; chemotherapeutic drugs, such as etoposides and teniposides, cytokines such as tumour necrosis factors and transforming growth factors; and stimulation of T-cell receptors.
When apoptosis is unregulated, disease results. Unregulated apoptosis is involved in diseases such as cancer, heart disease, neurodegenerative disorders, autoimmune disorders, and viral and bacterial infections. Cancer, for example, not only triggers cells to proliferate but also blocks apoptosis. Cancer is partly a failure of apoptosis: the orders for the cells to kill themselves by apoptosis are blocked. New cancer treatments that involve inducing apoptosis are being researched.
Disease and shock can cause cardiac cells to induce apoptosis. For example, cells deprived of oxygen after a heart attack release signals that induce apoptosis in cells in the heart. New treatments involving apoptosis blockers are being developed.
Apoptosis may also be involved in the destruction of neurons in people afflicted by strokes or neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and ALS (Lou Gehrig's disease). There is evidence suggesting that ischemia can kill neurons by inducing apoptosis. It has been shown that neurons that are resistant to apoptosis are also resistant to ischemic damage, thus, inhibition of apoptosis may be a therapeutic strategy for the treatment of neurodegenerative disorders such as stroke.
A failure of apoptosis in the immune system can lead to autoimmune diseases. T-cells differentiate between self and nonself (foreign) cells in the body. Autoimmune diseases such as rheumatoid arthritis, diabetes, and multiple sclerosis, result when a small percentage of T-cells attack the body's own tissue. Drugs are being developed that induce apoptosis in these T-cells.
Evidence also suggests that AIDs develops when the human immunodeficiency virus (HIV) sets off unregulated and untimely apoptosis in CD-4 and CD-8 cells, the defenders of the immune system.
There is an enormous therapeutic potential in controlling apoptosis in these diseases. Much research is now focussing on developing drugs that can either inhibit or induce apoptosis depending on the targeted disease. A major difficulty with researching apoptosis and drugs to control it is that a reliable marker of apoptosis has not yet been developed.
A marker is also needed in order to determine whether cells are dying or have been killed by apoptosis in the diagnosis of these diseases. For example, a marker for apoptosis could be used to determine the extend of neuronal damage caused by a stroke.
Apoptosis drugs are being used in therapy, and a reliable marker is needed in order to evaluate the progress of the therapy. For example, a major goal of some cancer chemotherapies has become to kill cancer cells by inducing apoptosis in these cells. It is estimated, however, that 45 percent of cancer drug treatments fail. It would be useful to have a method to assess the performance of new treatments in a reliable and effective manner. Currently, oncologists have to rely on manual measurements of tumour size and CAT Scan technologies to provide treatment feedback. The former is labour intensive while the latter is very expensive. In addition, both methods require at least one month of treatment to be effective. Furthermore, these methods do not indicate the nature of biochemical activities in the tumour. As such, there is a need for markers of apoptosis in order to determine whether apoptosis has been induced in tumour cells by the cancer chemotherapy.
Apoptosis is one type of cell death, another is necrosis. Cell death by necrosis often interferes with a determination of whether apoptosis has been induced. There are, however, distinguishing characteristics between the two. In contrast to apoptosis, necrosis is not genetically controlled, rather, it is induced by severe environmental trauma, such as chronic doses of heavy metals, chemicals, or extreme heat. The cells have little or no time to respond to the environmental stress and therefore die. Since RNA and protein synthesis are not required in order for cell death to occur, necrosis cannot be inhibited by drugs.
Necrosis is characterized by the swelling and rupturing of cells, the loss of membrane integrity, a random breakdown of DNA into fragments of variable size, and the phagocytosis of cellular debris by macrophages. The release of lysosomal enzymes damages neighbouring cells, thus, cells die in groups. This produces an inflammatory response in tissue. Cell death by necrosis involves no direct RNA or protein synthesis.
The differences in characteristics between apoptosis and necrosis allow one to differentiate between cell death by apoptosis and cell death by necrosis based on the following combinations of characteristics, apoptosis involves RNA and protein synthesis, the production of laddered DNA fragments, the maintenance of membrane integrity, and the exclusion of vital dyes during the process of dying by apoptosis; necrosis involves no RNA or protein synthesis, the production of random DNA fragments, the absence of membrane integrity, and the retention of vital dyes.
Despite the notable differences between apoptosis and necrosis, many long and tedious procedures are required to determine by which mechanism cell death has occurred. These procedures do not identify whether apoptosis is starting or is ongoing in a cell, this causes uncertainty in research, in the development of drugs that induce or inhibit apoptosis, in diagnosis, and in medical treatment. To date, a reliable marker for the early detection of apoptosis does not exist.
To date, a reliable marker for the early detection of apoptosis does not exist. Currently, the marker most commonly used to detect apoptosis is TUNEL labeling of the 3'-OH free end of DNA fragments produced during apoptosis (Gavrieli, Y. et al. (1992) J. Cell Bio. 119:493). The TUNEL method consists of catalytically adding a nucleotide, which has been conjugated to a chromogen system or a to a fluorescent tag, to the 3'-OH end of the 180-bp oligomer DNA fragments in order to detect the fragments. The presence of a DNA ladder of 180-bp oligomers is indicative of apoptosis.
Procedures to detect cell death based on the TUNEL method are offered by both Boehringer Mannheim (Cell Death Kit) and Oncor (Apoptag Plus). This method involves a number of limitations. Early detection of apoptosis is not possible with this method because the DNA ladder is an end-point in the apoptosis pathway. Also, although the TUNEL method distinguishes live cells from dead, it does not accurately determine whether the cells died by apoptosis or necrosis. False positives are often obtained when using the TUNEL method as a result of DNA fragments from cells that have died by necrosis: random DNA breakdown during necrosis generates DNA fragments that have 3'-OH ends. False negatives can also occur in certain cell types or situations where apoptosis does not lead to DNA laddering. Furthermore, the method is not quantitative since the amount of DNA fragments per cell is dependent upon the stage of apoptosis of the cell. In addition, this method is limited to use in flow cytometry and immunofluorescence, which renders it expensive and time consuming.
Another marker that is currently available is annexin, sold under the trademark APOPTEST.TM.. This marker is used in the "Apoptosis Detection Kit" offered by R&D Systems. During apoptosis, a cell membrane's phospholipid asymmetry changes such that the phospholipids are exposed on the outer membrane. Annexins are a homologous group of proteins that bind phospholipids in the presence of calcium. A second reagent, propidium iodide (PI), is a DNA binding fluorochrome. When a cell population is exposed to both reagents, apoptotic cells stain positive for annexin and negative for PL, necrotic cells stain positive for both, live cells stain negative for both. This marker, however, suffers from a number of problems. Annexin has a strong potential for a lack of specificity due to the fact that it is not antigenic. As well, its use is limited to cells grown in suspension, however, most cells are adherent and are grown on a matrix.
The method also requires the use of live or unpreserved cells. Furthermore, this method requires the use of a flow cytometer, expensive equipment that is not always readily available.
There is therefore a great need for a specific, antigenic, versatile marker for the rapid detection of cell death by apoptosis, which can be used for research, diagnostics, and therapeutics. This marker must be able to distinguish between cell death by apoptosis and cell death by necrosis.
The classical cell model to study apoptosis is the isolated thymocyte, a small lymphocyte in the cortex of the thymus. This model, however, includes a number of limitations as follows: apoptosis occurs too rapidly, making it difficult to delineate the sequence of events, thymocytes are predisposed to apoptosis, and thymocytes are non-adherent, and the role of extracellular matrix proteins (ECM) in apoptosis cannot be studied. For these reasons, an adherent cell model was developed from which cells at four different stages of apoptosis can be isolated and studied (Desjardins and MacManus (1995) Exp. Cell Res. 216:380-387).
Recently, it was reported that human and rodent cells undergoing apoptosis express high levels of a protein, termed apoptosis specific protein (ASP) (Grand et al., (1995) Exp. Cell Res. 218:439-451). Induction of apoptosis coincides with the expression of ASP. ASP is not detected or is detected in very low amounts in viable cells and cells dying passively by necrosis. It was concluded that ASP constitutes a powerful marker for the diagnosis and quantitation of apoptosis.
The protein GP46 is known in the literature as HSP47, HSP46, or colligin. This protein was first discovered by Kurkinen et al., (1984) J. Biol. Chem. 259:5915-5922, who described a 47 kDa glycoprotein from murine parietal endoderm cells that bound specifically to gelatin, collagen I, and collagen IV, hence, the name colligin. Later, similar glycoproteins were described in other cell types, including L6 rat myoblasts, 3T3 fibroblasts, keratinocytes, and chick embryo fibroblasts (Cates et al., (1984) J. Biol. Chem 259:2646-2650; Taylor et al., (1985) Exp. Cell Res. 159:47-54; Hughes et al. (1987) Eur. J. Biochem. 163:57-65; and Nagata et al., (1986) J. Cell. Biol. 103:223-229). The similarity between these proteins was later confirmed by comparing cDNA sequences between some of the cell lines (Clarke and Sanwal (1992) Biochim. Biophys. Acta 1129:246-248).
GP46 is localized in the lumen of the endoplasmic reticuhim (ER) of the cell (Nandan et al., (1988) Exp. Cell Res. 179:289-297; Saga et al., (1987) J. Cell Biol. 105:517-527). It has an RDEL (Arg-Asp-Glu-Leu) sequence at the C-terminus which serves as an ER retention signal (Pelham, (1990) Trends Biochem. Sci. 15:483-486). GP46 is known to bind to denatured collagen, collagen I, collagen IV, and procollagen I. Its role may be to act as a molecular chaperone by assisting in protein folding of collagen during its synthesis (Jain et al. (1994) Biochem. J. 304:61-68; Nakai et al., (1992) J. Cell Biol. 117:903-914). It is a member of the serine-proteinase inhibitor family (serpin) and may protect procollagen I chains from degradation by inhibiting collagenase in the ER (Jain et al., (1994) Biochem. J. 304:61-68; Clarke et al., (1993) J. Cell Biol. 121:193-199).
A number of findings have linked the GP46 protein to apoptosis. It has been observed that GP46 mRNA increases in neuronal cells during apoptosis, and that the GP46 levels remain high for 48 hours (Higashi et al., (1994) Brain Res. 650:239-248). As well, it has been reported that when anti-sense RNA of GP46 is added to cells, apoptosis is induced and the cells die (Nagata et al., (1995) "Regulation and Function of Collagen-Specific Molecular Chaperone-Like Protein HSP47" in J. Cell. Biochem. Suppl. 190:195 and oral presentation at the Keystone Symposia on Sress Proteins in March, 1995).
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. Moreover, publications referred to in the following discussion are hereby incorporated by reference in their entireties in this application.